Chapter title |
Allosteric Regulation of Human Liver Pyruvate Kinase by Peptides that Mimic the Phosphorylated/Dephosphorylated N-Terminus.
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Chapter number | 18 |
Book title |
Allostery
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Published in |
Methods in molecular biology, November 2011
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DOI | 10.1007/978-1-61779-334-9_18 |
Pubmed ID | |
Book ISBNs |
978-1-61779-333-2, 978-1-61779-334-9
|
Authors |
Charulata B. Prasannan, Qingling Tang, Aron W. Fenton |
Abstract |
An advantage of studying allosteric regulation over covalent modification is that allostery allows the experimentalist to vary the concentration of effector, thereby allowing independent quantification of effector binding and allosteric coupling. In turn, this capacity allows the use of effector analogues to determine which regions of the effector contribute to effector binding and which contribute to allosteric regulation. Like many other proteins, human liver pyruvate kinase (hL-PYK) is regulated by phosphorylation. The phosphorylation of hL-PYK occurs on Ser12 of the N-terminus. Phosphorylation appears to interrupt an interaction (distant from the active site) between the N-terminus and the main body of the protein. Since this interaction increases the affinity of hL-PYK for the substrate (phosphoenolpyruvate, PEP), phosphorylation-dependent interruption of the N-terminus/main-body interaction results in an antagonism of PEP binding. Due to the advantages of studying an allosteric system, we detail a protocol to express and purify N-terminal peptides of hL-PYK using a SUMO-fusion system. We further demonstrate that these peptides act as allosteric regulators that modulate the affinity of hL-PYK for PEP. |
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